Arbutin (hydroquinone glucoside) and its aglycone, hydroquinone, are compounds used as skin whitening agents. Skin pigmentation is mainly determined by the amount of melanin produced by melanocytes. The biosynthetic pathway leading to melanin formation begins with the conversion of tyrosine to dihydroxyphenylalanine (DOPA) via the enzyme tyrosinase (EC 1.14.18.1). Inhibition of tyrosinase decreases the amount of melanin produced by the melanocytes, leading to the depigmentation of the skin. Arbutin and monoester derivatives of arbutin have been shown to decrease the amount of melanin produced by melanocytes by inhibiting tyrosinase (U.S. Pat. No. 6,306,376).
Hydroquinone and derivatives of hydroquinone, such as hydroquinone ethers, are also used as depigmenting agents. However, negative side effects from the use of these compounds have been reported. These compounds are particularly irritating and cytotoxic to melanocytes (U.S. Pat. No. 6,306,376). The use of arbutin as a depigmenting agent is preferred over hydroquinone because of arbutin's reduced toxicity in comparison to the aglycone.
Arbutin has also been reported to be useful as an antioxidant, an antimicrobial agent, an anti-inflammatory agent, and possibly as an inhibitor of carcinogenesis (melanoma). However, arbutin's commercial use has been limited due to its high cost. No low-cost commercial production route to this chemical exists. Current methods for production include chemical synthesis (U.S. Pat. No. 3,201,385; U.S. Pat. No. 6,388,103; and JP 62-226974A); extraction from plants naturally producing arbutin; bio-transformation using either microbial hosts or plant cell cultures, seeds, or seedlings expressing suitable glucosyltransferases contacted with hydroquinone and UDP-glucose (Arend et al., Phytochemistry, 53: 187–193 (2000); Arend et al., Biotech. Bioeng., 76(2):126–131 (2001); Hefner et al., Bioorg. Med. Chem., 10: 1731–1741 (2002); and JP 07224083A), or by a similar process where a mixture of glucose and hydroquinone is contacted with a β-glucosidase (JP 05176785A).
Chemical synthesis is not a cost-effective way to produce arbutin. Chemical synthesis methods usually require expensive starting materials, various expensive and toxic solvents and catalysts, significant energy input, and a subsequent purification step to remove impurities. The investment in non-renewable resources is both expensive and environmentally unfriendly.
Many higher plants naturally produce arbutin. Members of the Eracaceae, Rosaceae, and Saxifragaceae families have been reported to produce arbutin in amounts up to 20% dry weight (leaf). However, these plants suffer from poor agronomic performance (Arend et al., Phytochemistry, 53:187–193 (2000)). Cost-effective production of arbutin in plants requires a crop plant species with high agronomic performance and an established processing infrastructure.
Even though various routes to the production of arbutin exist, the possibility of using green plants with high agronomic performance for the commercial production of chemicals has become an increasingly attractive alternative. As opposed to organic synthesis, green plants constitute a renewable energy source. Because of their unique photosynthetic capability, the only raw materials that are required to produce carbon-based compounds in green plants are carbon dioxide, water and soil, with sunlight providing the ultimate energy source. In comparison to existing fermentation facilities that are limited in size, green plants constitute a huge available biomass that could easily accommodate large-scale production of chemicals, even those requiring high-volume, low-cost applications.
Even though in vivo plant production offers a larger potential biomass, microbial production is also an attractive alternative in comparison to expensive, energy-intensive, and environmentally unfriendly chemical synthesis. Industrial microbes can be genetically modified to produce the desired genetic end product. Hefner et al. (Bioorg. Med. Chem., 10:1731–1741 (2002)) produced arbutin by exogenously supplying hydroquinone, a toxic and expensive substrate, to either cell suspension cultures of Rauvolfia serpentina expressing an endogenous hydroquinone glucosyltransferase (HQ GT) or to recombinant E. coli cells expressing the same enzyme. However, suitable recombinant microorganisms modified to produce commercially useful levels of arbutin currently do not exist.
Ran et al. (J. Am. Chem. Soc., 123:10927–10934 (2001)) teach a method to produce hydroquinone using a microbial catalyst to convert glucose to the intermediate quinic acid. The quinic acid is subsequently isolated and chemically converted into hydroquinone. Ran et al. (supra) postulate that glucose could theoretically be converted into hydroquinone via a pHBA intermediate using the pHBA 1-hydroxylase activity of Candida parapsilosis. However, Ran et al. do not teach the isolating of the pHBA 1-hydroxylase gene, the sequence of the gene, nor a method to produce arbutin in plants and microbes.
The problem to be solved therefore is the lack of methods and materials to produce arbutin (hydroquinone glucoside) in transgenic plants, in microorganisms, or in vitro at commercially-useful levels.